X-ray imaging technology plays a critical and important role in healthcare and biomedical research. X-ray radiography is one of the most important and widely deployed medical diagnostic methods that can be traced back to the invention of x-rays more than 100 years ago. Recently, soft x-ray cryo-microscopy has been demonstrated to offer non-destructive high resolution three dimensional tomographic imaging of single biological cells with a resolution of about 25 nanometers (nm) in two dimensions (2-D) and 60 nm in three dimensions (3-D). Important trace elements in a biological specimen such as a cell or tissue can be mapped with sensitivity better than parts per billion at a spatial resolution of about 100 nm using a synchrotron based x-ray fluorescence microscope.
An additional important application for higher performance zone plate lenses is high throughput laboratory protein crystallography systems. The knowledge of three-dimensional structures of proteins gives insight into their functionality, and can provide key information for rational drug design to treat human diseases. The underlying hypothesis is that if the structure of the active site of a critical enzyme in a metabolic or regulatory pathway is known, the chemical compounds can then be designed to inhibit or affect the behavior of that specific enzyme. Information on the structural design of proteins also facilitates protein engineering to purposely modify the structures of proteins for specific applications. Thus far, structures for about 19,000 proteins have been deposited in the protein data bank, including 3300 entries added in 2001, out of about 100,000 proteins assumed to be manufactured in the human body, and of course protein structures from other organisms are of great scientific, societal, and commercial interest as well.
At present, it is very difficult to predict the proper folded structure for proteins based on their amino acid sequences. X-ray protein crystallography is the single most useful tool to determine macromolecule structures such as proteins, especially for molecular weights above about 30 kD. Currently, a typical laboratory based x-ray protein crystallography facility is expensive to equip, and it takes a day or two to collect diffraction data sufficient to determine a new protein structure once proper crystallization conditions have been found. Considering the possibility that many attempts are often made to obtain data sufficient for structural determination (due to sample quality, etc.), solving structures of all 10,000 proteins will be a great challenge in both facility cost and human effort. The alternative approach is to use synchrotron beamlines for data collection, where the higher flux and brightness of the source, along with its energy tunability for methods like multiple wavelength anomalous dispersion (MAD), means that an entire dataset appropriate for rapid structure determination can be collected in a matter of hours.
More generally, x-ray microscopy is a complementary tool to light and electron microscopes for biomedical and other research. In recent years, increasingly powerful imaging methods have provided more detailed views of cells. Making use of the development of function specific labeling and contrast enhancement techniques, many forms of imaging techniques have been employed to enable researchers to make key discoveries of the working mechanisms of cells and biological systems. The ability to perform function specific imaging using labels, such as green fluorescent protein, quantum dots, and immunogold, provides the means to elucidate the important link between the working mechanism of cells and tissues and molecular machines at the protein, DNA, and macromolecular assembly level. Optical and electron microscopes are essential for many of these important discoveries and have been widely deployed in biomedical research laboratories. However, various limitations exist in the current microscopy techniques. Existing systems can not address the needs of biologists who wish to visualize the organization of organelles inside a single cell, or connections (such as synapses) between two or more cells with high resolution.
Critical to the development and performance of x-ray microscopes are x-ray lenses that image the x-rays from the object of interest onto a detector system such as an optical stage or directly on an electronic detector device. The task of focusing x-rays has occupied physicists for over a century. All three well known optical phenomena (refraction, reflection, and diffraction) have been exploited to produce x-ray lenses with unique advantages and limitations.
The main challenge (but also a unique advantage) when dealing with x-rays is that they interact with matter only very weakly. The difference in the refractive index between vacuum (or air) and solids is less than 10−5 for 8 keV x-rays even for dense materials like gold. To overcome this, a concept of using many weakly focusing lenses arranged along the optical axis (termed “compound refractive lens”) was proposed about 10 years ago to increase the effective numerical aperture of refractive x-ray lenses. Although significant progress has been made over the years, its performance in terms of numerical aperture and resolution significantly lags behind the currently available reflection and diffraction based x-ray lenses. The numerical aperture achievable using this type of x-ray lenses is fundamentally limited due to photoelectric absorption for low energy x-rays and Compton scattering for high energy x-rays.
Reflection based lenses are capable of obtaining fairly large numerical apertures especially when the reflecting surface is coated with a multilayer with a small period, but their resolution is limited by stringent tolerance requirements in the smoothness and the slope error of the reflecting surface. Furthermore, the field of view of a reflection based lens is typically very small unless a special, axially symmetric optical design such as a Wolter mirror is used, which employs two consecutive reflections off internal surfaces of parabolic and hyperbolic shape. Since it is difficult to achieve 100 nm resolution using a single monolithic reflecting surface under the optimal conditions, no one has been able to produce a reflection based lens with a reasonable field of view and sub-micrometer resolution.
X-ray zone plates are diffraction based lenses and currently offer the best optical performance for high resolution x-ray imaging and home-lab protein crystallography utilizing a microfocus x-ray source. They combine the highest spatial resolution (˜20 nm with soft x-rays) achievable over the whole electromagnetic spectrum and a large field of view that can be up to ⅓ of the zone plate diameter (typically many tens of micrometers).
Currently the best performing zone plate lenses for x-rays in terms of resolution and efficiency are fabricated by means of a deep pattern transfer process based on semiconductor/microelectromechanical system (MEMS) technology. In this process a zone plate pattern is written by electron beam lithography in a very thin layer of high-resolution photoresist. This pattern is transferred by a directional (anisotropic) reactive ion etch into a thick layer of photoresist, which forms the mold for electrochemical plating of a metal. For soft x-rays (<1 keV) zone plates with outermost zone widths as small as 20 nm have been demonstrated. For hard x-rays the zone thickness requirement increases drastically (from 100 nm for soft x-rays to 1600 nm for 10 keV x-rays) making the fabrication of fine outermost zones much more challenging. Current state of the art zone plate fabrication technology is based on a multi-level lithographic process based on microfabrication technology for manufacturing semiconductor devices. The process however is limited in the smallest achievable zone width and especially in obtaining adequate thickness for efficient focusing of multi-keV x-rays.